Critical Variables affecting clinical-grade production of the self-inactivating gamma-retroviral vector for the treatment of X-linked severe combined immunodeficiency

J. C.M. Van Der Loo, W. P. Swaney, E. Grassman, A. Terwilliger, T. Higashimoto, A. Schambach, S. Hacein-Bey-Abina, D. L. Nordling, M. Cavazzana-Calvo, A. J. Thrasher, D. A. Williams, L. Reeves, P. Malik

Research output: Contribution to journalArticle

11 Scopus citations

Abstract

Patients with X-linked severe combined immunodeficiency (SCID-X1) were successfully cured following gene therapy with a gamma-retroviral vector (gRV) expressing the common gamma chain of the interleukin-2 receptor (IL2RG). However, 5 of 20 patients developed leukemia from activation of cellular proto-oncogenes by viral enhancers in the long-terminal repeats (LTR) of the integrated vector. These events prompted the design of a gRV vector with self-inactivating (SIN) LTRs to enhance vector safety. Herein we report on the production of a clinical-grade SIN IL2RG gRV pseudotyped with the Gibbon Ape Leukemia Virus envelope for a new gene therapy trial for SCID-X1, and highlight variables that were found to be critical for transfection-based large-scale SIN gRV production. Successful clinical production required careful selection of culture medium without pre-added glutamine, reduced exposure of packaging cells to cell-dissociation enzyme, and presence of cations in wash buffer. The clinical vector was high titer; transduced 68-70% normal human CD34 + cells, as determined by colony-forming unit assays and by xenotransplantation in immunodeficient NOD.CB17-Prkdc scid/J (nonobese diabetic/severe combined immunodeficiency (NOD/SCID)) and NOD.Cg-Prkdc scid Il2rg tm1Wjl/SzJ (NOD/SCID gamma (NSG))) mice; and resulted in the production of T cells in vitro from human SCID-X1 CD34 +cells. The vector was certified and released for the treatment of SCID-X1 in a multi-center international phase I/II trial.

Original languageEnglish (US)
Pages (from-to)872-876
Number of pages5
JournalGene Therapy
Volume19
Issue number8
DOIs
StatePublished - Aug 1 2012

Keywords

  • clinical-grade
  • gamma-retrovirus
  • GMP
  • process development
  • SCID-X1
  • self-inactivating

ASJC Scopus subject areas

  • Molecular Medicine
  • Molecular Biology
  • Genetics

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    Van Der Loo, J. C. M., Swaney, W. P., Grassman, E., Terwilliger, A., Higashimoto, T., Schambach, A., Hacein-Bey-Abina, S., Nordling, D. L., Cavazzana-Calvo, M., Thrasher, A. J., Williams, D. A., Reeves, L., & Malik, P. (2012). Critical Variables affecting clinical-grade production of the self-inactivating gamma-retroviral vector for the treatment of X-linked severe combined immunodeficiency. Gene Therapy, 19(8), 872-876. https://doi.org/10.1038/gt.2012.37